Project 2 Aim 1. To investigate biologic consequences of PI-3Kinase/AKT activation on NB tumor biology Inhibition of AKT pathway in neuroblastoma inhibits tumor cell growth in vitro and in vivo. Our previous studies have identified activation of the PI3Kinase/Akt/GSK3 pathway mediates resistance to chemotherapy in neuroblastoma cells. Moreover our genetic and pharmacologic studies indicated that activation of AKT alone attenuated the effects of chemotherapy in neuroblastoma cells. Since activated AKT is more highly expressed in tumors of Neuroblastoma patients with a poor prognosis, we screened a number of inhibitors of the AKT that could enhance the efficacy of chemotherapy in our pre-clinical in vitro models. Studies have shown that it is reasonable to pursue inhibition of the TrkB receptor as an adjunct to therapy, the use of an Akt inhibitor would be valuable in combination or following receptor targeted therapy since targeting receptors may cause activation of AKT due to feedback suppression. Moreover, targeting AKT inhibits a key survival signaling node used not only by TrkB but by other membrane receptors shown to enhance NB cell survival such as IGF1R, IL-6 receptors and VEGFR. By targeting a common downstream signal intermediary, one covers a broader spectrum of signaling pathways that use this common intermediary to affect biologic function. 1. Studies on AKT PH domain inhibitor Perifosine. We focused on the AKT inhibitor, Perifosine, because it is Phase I/II trials in adult cancers, with a toxicity profile that is controllable with systemic therapy. Perifosine was tested in a number of our pre-clinical in vitro models as well as our in vivo murine heterotypic and orthotopic xenograft models. Perifosine inhibits activation of AKT and more impressively inhibits tumor cell growth in all 4 cell line models tested in vitro and in vivo. In the AS neuroblastoma model system there was complete tumor regression. In the least sensitive model, the addition of chemotherapy with Perifosine caused dramatic tumor regressions. Moreover Perifosine inhibited the growth of NB tumors containing ALK mutations, even those with the ALKF1174L mutation, which are less sensitive to some small molecule inhibitors of ALK. Recently the Pediatric Phase I study of Perifosine showed activity in neuroblastoma. Based on our pre-clinical study and the Phase I results the FDA approved Perifosine for Orphan Drug Status for the treatment of Neuroblastoma. 2. Studies on AKT allosteric inhibitor MK2206. While we initiated studies using Perifosine as an AKT inhibitor, we knew Akt was not its only target. To more directly assess the contribution of AKT to our models, we established an MTA with Merck to study their AKT inhibitor MK2206. This is an allosteric AKT inhibitor which acts by specifically binding to the PH domain of AKT thus preventing its binding to phosphatidylinositol-3,4,5-trisphosphate. There is differential inhibition of AKTisoforms; IC50-Akt1=5.3nM, -Akt2=12nM and -Akt3=65nM. We find MK2206 inhibits AKT activity as assessed by inhibition of phosphorylation of downstream target S6 kinase in the pharmacologic range but the growth of only 3/9 NB cell lines tested is inhibited within this range. In this study, we investigated whether a novel allosteric Akt inhibitor MK-2206 increased the sensitivity of NB to etoposide or rapamycin. Here we showed that in vitro, a synergistic effect was detected in combination of MK-2206 with etoposide through apoptosis, and MK-2206 enhanced the sensitivity to rapamycin via reactive oxygen species; in vivo, a significant increased anti-tumor growth effect and murine survival advantage were observed in the combination of MK-2206 with etoposide or rapamycin. This study provides the basis for the combination use of molecular targeted drug of Akt with other treatment regimens in NB or other cancers with aberrant Akt activation and has important clinical implications. These studies provide proof of principle that targeting Akt alone will inhibit neuroblastoma cell growth and will synergize with cytotoxics such as etoposide or mTOR inhibitors. Specific Aim 2. To investigate STAT3 activation and NB tumor biology In a recently published study, we evaluated the involvement of aberrantly activated IL-6/JAK/STAT pathway in Neuroblastoma, Rhabdomyosarcoma and Ewings Sarcoma. 1. Studies evaluating JAK/STAT3 inihbitor AZD1480. We identified that this pathway was activated in these pediatric tumors and could be inhibited using a JAK1/2 selective inhibitor AZD1480. Our data indicate that AZD1480 blocks the endogenous constitutive as well as induced STAT3 activation. AZD1480 treatment decreased cell viability in 7/7NB, 7/7RMS and 2/2 EWS cell lines tested (median EC50 is 1.5uM, ranging from 0.36-5.37uM). AZD1480 induced cell growth inhibition and apoptosis in vitro, along with decreased expression of direct STAT3 targets, including cell cycle regulators CyclinD1, 3 and CDC25A, anti-apoptotic proteins Bcl-2 and survivin, metastasis-related factor TIMP-1 and oncogene c-Myc. AZD1480 treatment increased caspase3/7 activity that could be rescued by a pan-caspase inhibitor indicating AZD1480 induced apoptosis was caspase-dependent. In vivo studies showed significantly decreased tumor growth and prolonged overall survival in AZD1480-treated tumor-bearing mice compared with controls. Tumors from AZD1480 treated mice showed inhibition of STAT3 activation as well as its downstream targets. Our study provided the first preclinical evaluation of the anti-tumor potency of JAK inhibitor AZD1480 in pediatric solid tumors. Demonstration of in vitro and in vivo anti-tumor activity of AZD1480 provides and extends the rationale for the clinical evaluation of AZD1480 in the treatment of pediatric patients with solid malignancies. A limitation of this study is the off-target effects of AZD1480 and toxicity in the Phase I study led to discontuation of the development of this drug. 2. Evaluation of genetic inhibition of STAT3. We have continued pre-clinical studies evaluating genetic inhibition of STAT3 using tet-regulated shSTAT3 expression vectors. These studies are complemented by ones in which we will evaluate a first in class siRNA targeting STAT3.